- Proteins and Peptides
- Lysates and Cell Lines
1. Prepare cell / tissue lysate. Make up lysates to a total volume of 10-20ul with 4x SDS sample buffer containing a reducing agent (either 10% B-ME or 0.3M DTT in the 4xSB).
2. Prepare SDS-PAGE gel. If pouring your own, this should be performed before starting the sample preparation. Numerous manufacturers sell a variety of pre-cast gel types and polyacrylamide concentrations with shelf life in the region of a year (Invitrogen, Bio-Rad) and a corresponding sample buffer, running buffer and denaturant to go with this. This alternative to preparing your own is a time saving way. When considering what percentage gel to use, this depends on the protein of interest. For proteins whose MW is over 100kDa, the percentage is 7%, for 50-100kDa, it is 10%, for 20-50kDa, it is 12%, and it is 15% for 3. Place gel in running chamber and fill with 1x SDS Running Buffer. Use 10x stock.
4. Heat samples at 95-100? for 5 minutes.
5. Load 5ul of a control pre-stained protein ladder to track sample migration in the first lane of the gel. This is also useful for the transfer step to verify the procedure is successful. These are widely available for most suppliers (Invitrogen, Bio-Rad, Fermentas, etc.).
6. Load boiled samples. Electrophorese at constant 100V until the blue bromophenol dye reaches the bottom of the gel.
1. Place gel and blotting pads in transfer buffer solution.
2. Transfer gel to filter paper wetted in transfer buffer solution.
3. Assemble transfer sandwich by orientating cathode, filter paper, gel, membrane (nitrocellulose), filter paper, anode so protein transfer goes in the direction of cathode to anode. The cathode is typically coloured black and the annode red. Electrophorese for 90 minutes at room temperature at 100V or overnight at 4? at 20V. Proceed with electrophoresis on ice or in a cold room as the transfer generates a lot of heat!
4. Check membrane for transfer of control protein ladder.
1. Incubate membrane in blocking solution for 1 hour at room temperature or at 4? overnight with shaking. The blocking solution is normally composed of 5% non-fat milk in TBS-T, and some antibodies require BSA in place of milk. To make TBS-T, using 10x TBS, add Tween-20 to a final concentration of 0.05%.
2. Incubate primary antibody overnight at 4? or at room temperature for 2 hours prepared in 5% milk/TBST.
3. Remove antibody solution and wash membrane three times for 5-15 minutes in TBST.
4. Incubate membrane with an appropriate secondary antibody (peroxidase conjugated) for 1 hour at room temperature in 5% milk/TBST. For example if your primary antibody was raised in rabbit, a goat anti-rabbit HRP should be used as a secondary antibody.
5. Remove antibody solution and wash membrane three times for 5-15 minutes in TBST.
Use chemiluminescence for detection. Expose membrane to X-ray film for 1 minute to 1 hour, depending on signal intensity.